Vaccination has proven to be an effective way to prevent various diseases. Today, vaccines are available against many human pathogens, including bacteria, viruses, parasites, and fungi, and new vaccines are constantly being developed.
Improvements to vaccine technology have focused on development of various antigens to enhance immunogenicity and inducibility for both cell-mediated immune responses and humoral immune responses. For example, for candidate antigens such as small protein subunits or peptides having low immunogenicity, peptide aggregates and aggregates of linked peptides have been tested as antigens for subunit vaccines. Linked peptide antigens currently being evaluated in clinical trials include polymers of linked peptides from group A streptococcus as a vaccine against rheumatic fever (Brandt et al 2000) and a plasmodium peptide polymer to induce a strong immune response against malaria (Nardin et al 2000).
Vaccination also can be used for treating noncommunicable diseases. For example, immunotherapy has been proposed to treat autoimmune diseases, viral infection-induced cancers, endogenous tumor antigens-induced cancers, and Alzheimer's Disease. The general concept is to generate strong and persistent cytotoxic-T-cell responses against various disease-associated antigens by immunizing an individual with disease-associated antigens and/or antibodies against the disease-associated antigens before (to prevent) or after (to treat) occurrence of the disease.
When developing vaccines, increasing the total amount of antigen has proven to be problematic. Preparation of high potency antigens is needed to minimize the total antigen content of the vaccines. It has been shown that some antigens that have poor immunogenicity in monomer form were able to induce the production of antibody once they were cross-linked. For example, chemically cross-linked pertussis antigens have induced higher immune response than monomeric pertussis antigens. (Watanabe et al, 2001). Despite this advantage, however, chemical cross-linking typically involves a toxic cross-linking agent, such as glutaraldehyde, glyoxal, and octansdialdehyde.
Cross-linked protein crystals also have been proposed for use as safe and stable antigens. However, protein crystals are also prepared by chemical cross-linking and, further, have to be available in large quantities that require cumbersome manufacturing processes (Clair et al, 1999).
Another drawback to improving vaccine technology through cross-linking is that biological agents have limitations in that they are very substrate specific; i.e., such agents react with only a limited number of compounds. Accordingly, biological agents have not been used as cross-linking agents for preparing antigens or in other immunological applications. Most known cross-linking biological agents such as enzymes or peptides are not desirable for use because of difficulties in providing an adequate quantity, high cost, and difficulty in purification of the agents. For example, cross-linking biological agents such as microbial transglutaminase has been purified mainly from culture medium of the extra-cellular microbial transglutaminase. Such preparation is adequate for the application of transglutaminases for cross-linking in industrial applications. However, microbial transglutaminases purified from crude lysate or culture medium of batch fermentation may not be suitable for vaccine development and may include toxic compounds and contaminating proteins which could induce undesirable cross-reactive antibodies.
Thus, although various biological agents such as enzymes have been employed in limited cross-linking applications, such as the preparation of foods or cosmetics, methods for cross-linking a broad scope of proteins or peptides, particularly for therapeutic applications, currently do not exist. Therefore, there exists a need for methods of producing biological agents which catalyze cross-linking on a broad scope of protein substrates. Such agents may be used, e.g., to cross-link proteins to be used as antigens for immunotherapy and antibody production.